1. Field of the Invention
The field of this invention relates generally to proteins (convertases) which cleave precursor molecules in order to convert them to various active forms. Molecules which require such processing include hormone and neuropeptide precursors (neuromodulators). More specifically, this invention concerns a family of endoproteases structurally related to the following: kex2-endoprotease of yeast, bacterial subtilisins, and the human fur gene product, furin. Specifically, this invention relates to convertins, novel mammalian proteases and more specifically in this family convertin I. This invention is also related to the nucleic acids coding for this family of proteins, and to their complementary nucleic acid sequences. Methods of producing this family of proteins, for example, by use of human insulinoma derived cDNA or other methods of genetic engineering, are also provided. Applications and advantages of the protein family are presented, more specifically those of convertin I.
2. Description of Related Art
A. Maturation of Proteins PA0 B. Yeast Models PA0 C. A Processing Enzyme in Fish PA0 D. Mammalian Processing Enzymes PA0 E. Complexities of Processing PA0 F. Bacterial Subtilisins PA0 G. The Fur Gene Product, Furin
Although there are many kinds of regulatory peptides which differ in their function, cellular localization, and structure, they share a common property in that they are almost always initially synthesized in a larger form, that is, a precursor form, and subsequently are processed to form biologically active products. The first example of this processing was worked out for proinsulin, a larger precursor form of insulin, by Steiner, et al. (1976). The terminology developed by Steiner and colleagues to describe this phenomenon in terms of the intermediates produced during maturation, have been adapted to various intermediates produced during the biosynthesis of other regulatory peptides.
Regulatory peptides such as hormones and neuropeptides must undergo maturation processes to become biologically active, including proteolytic processing by various endoproteases. These endoproteases can cleave at various adjacent amino acid residues. As described in a review by Andrews et al. (1987), there are also enzymes involved in proteolytic cleavage to remove the hydrophobic signal peptide located at the amino terminus of the precursor. The "signal hypothesis" model of protein export from the cell was developed by Blobel and Dobberstein in 1975. Preproinsulin was demonstrated by Chan, et al. in 1976. An example of experiments which demonstrated the cleavage of the signal sequence are those of Minth, et al. (1984) using human pheochromocytoma, and those that isolated and characterized a 37 kd protease which is capable of cleaving the signal proteins of preproproteins from the honey bee prepromellitin, the human preproplacental lactogen, and preproinsulin. (Wickner, et al., 1985) This processing of "prepro-" forms of hormones and other secreted proteins occurs in the rough endoplasmic reticulum and results in removal of N-terminal segments of these proteins, depicted in FIG. I. There is subsequent cleavage of the "pro-" forms to release active products at single and double adjacent basic residues, followed by removal of the basic residues located at the C-terminus of the peptides by carboxypeptidase B-like enzymes. Proteolytic processing at dibasic amino acids represents an important step in the maturation of a large number of prohormones, neuropeptides, and other biologically active peptides and proteins.
While the synthesis of proteins which will eventually be secreted from the cell is usually initiated on cytoplasmic ribosomes, these very rapidly become tightly bound to the membrane of the endoplasmic reticulum across which the nascent peptide chain is transferred. Both eukaryotic and prokaryotic cells appear to use similar mechanisms for protein export. Bacterial cells can be used to export some eukaryotic proteins, and conversely, prohormones like proinsulin also can be processed by heterologous endocrine cells (Moore et al., 1983) and in distantly related eukaryotes such as yeast. (Thim et al., 1986). Bacteria can make, but do not cleave, prohormones, to yield the active forms.
In the yeast, a eukaryote, precursors for at least two biologically active peptides have been identified: pro a factor and the prokiller toxin (see reviews in Mizuno et al., 1988; Andrews et al, 1987). Yeast cells exist which are defective in the proteolytic processing of precursors of these two proteins to a mature form e.g. KEX2 defective strains. The KEX2 gene encodes a novel endoprotease which is specific for cleaving these two precursor substrates on the carboxyl side of pairs of basic residues. The KEX2 gene has been cloned and introduced into multi-copy plasmids so that the protease is over-produced. From these cells, sufficient quantities of the endoprotease were obtained to allow purification to reportedly about 100 fold. The catalytic properties have been determined, indicating that the substrate site preferences of the kex2 endoprotease are on the carboxyl side of arginine-arginine (ArgArg) and lysine-arginine (LysArg). Enzyme activity is calcium dependent, similar to mammalian proteases called calpains, but they differ in catalytic mechanism in that kex2 is a serine protease. The complete amino acid sequence of the KEX2-encoded protein, deduced from nucleotide sequencing by Mizuno et al. (1988), revealed extensive homology between the amino acid sequences of the catalytic domain of the kex2 protein and those of the bacterial subtilisins (Section F). Sequence similarities around the active site residues resulting from similarities in gene structures (FIG. 2) suggest an evolutionary relationship between these proteases.
Matsuo et al. (1985, 1987) have patented a 43 kd serine-type protease prepared from Saccharomyces cerevisae. This enzyme hydrolytically cleaves peptide bonds between two adjacent basic amino acids, and in vitro, converts prohormones to active forms. (U.S. Pat. Nos. 4,650,763, 4,704,1359, EP O158981A2). This enzyme was believed by the inventors to differ from that reported by Julius et al., (1984) (isolation of a gene (KEX2) encoding a yeast dibasic cleaving endoprotease). The inventors also claimed that the enzyme disclosed differed from the proteases reported by Fletcher et al., 1981; Loh et al., 1982; 1983; Mizuno et al., 1988. Cleavage was on the carboxyl side of repeating-X-Ala sequences, using enkephalin as a substrate. Subsequent work showed that kex2 showed no relation to this enzyme, as originally reported by Mizuno et al. in 1984 (Fuller, et al., 1988) and it is unlikely to be involved in precursor processing.
A protease has been identified from the anglerfish pancreatic islets by Fletcher, et al. (1981) and proposed to be one of the enzymes that processes proinsulin.
"The yeast KEX2 protease is the only enzyme that has a proven role in the activation of polypeptide hormones through cleavage at parts of basic residues. The enzyme that fulfills this role in higher eukaryotes has yet to be unequivocally identified," (Brennan and Peach, 1988) although various candidates have appeared on the scene, as discussed below.
An enzyme has been identified by Loh which converts mouse proopiomelanocortin to various products. This is a 70 kilodalton glycoprotein which has been purified from secretory vesicles of the bovine pituitary. However, it is unknown whether it functions in prohormone conversion in vivo. (Loh et al., 1987)
A prohormone converting enzyme which is found in rat microsomes and secretory granules has been described by Noe, et al. (1984) and said to be associated with membranes in the rat anterior pituitary neurointermediate lobe and rat hypothalamic synaptosomes. Noe suggested that the newly synthesized islet prohormones are membrane associated in the microsome and secretory granules and that the RER/Golgi complex and secretory granule membranes act as a matrix, uniting the enzyme and substrate. However, conclusive experimental evidence for this is lacking.
Davidson et al. (1988) reported the presence of two distinct Ca-dependent acidic endoproteases in lysates of secretory granules from a rat insulinoma. Type I cleaved on the C-terminal side of Arg 31, Arg 32 (B-chain/C peptide junction); type II, the C-terminal side of Lys 64, Arg 65 (C-peptide/A-chain function). Type II was postulated to also be active in the Golgi apparatus because of its more neutral pH optimum which allowed significant activity at pH 7.0 (the supposed pH of the Golgi). These results suggest that more than one processing enzyme may exist and this could be relevant to alternative processing. (Section E).
Proteolytic processing of the same precursor molecule may occur in different ways, perhaps providing greater flexibility for the organism. The reasons for alternative processing are unclear and the systems are complex. The main physiologically active peptide produced as a result of proglucagon processing in the pancreas is the hormone glucagon. However, a larger peptide is derived from the C-terminal portion of proglucagon which has not yet been shown to possess biological activity. In contrast, in certain cells in the gut, four different physiologically active proteins are processed from proglucagon. Thus, there may be multiple recognition sites for different converting enzymes to produce different molecules from the same precursor.
On the other hand, some endoproteolytic enzymes, like kex2, seem to have low specificity and can recognize and cleave several precursor proteins, even those from unrelated eukaryote species. (Thomas and Thorne, 1988.)
Subtilisins belong to a serine protease superfamily. Bacteria of the bacilli species secrete at least two distinct levels of extracellular protease: a neutral metalloprotease, and an alkaline protease which is functionally a serine endopeptidase, "subtilisin." Secretion of these proteases has been linked to the bacterial growth cycle, with greatest expression of protease during the stationary phase when sporulation also occurs. (Joliffe, et al., 1980; Hastrup et al., 1989).
A wide variety of subtilisins have been identified, and the amino acid sequences of at least 8 have been determined. Some have been cloned. Subtilisins are well characterized physically and chemically. In addition to knowledge of the primary structure (the amino acid sequence), over 50 high resolution X-ray structures of subtilisin have been determined which delineate the binding of substrate, transition state products, different protease inhibitors, and structural consequences for natural variation. (Review in Hastrup, et al., 1989). Subtilisins have found utility in industry, particularly in detergent formulations, because they are useful for removing proteinaceous stains. Determination of the relationship between the primary structure of subtilisin and its physical properties have revealed the significance of the methionine 222 residue as well as the amino acids functional in the active site, that is, aspartic acid 32, histidine 64 and serine 221. Asparagine 155 and serine 221 are within the oxyanion binding site. Mutations of these positions are likely to diminish proteolytic activity. (Hastrup, et al., 1989). Desirable commercial characteristics of subtilisins produced by mutagenesis include improved stability to oxidation, increased proteolytic ability or improved washability (stability during commercial use).
"The fur gene was discovered fortuitously by comparing inserts of certain cosmid clones encoding the fes/fps oncogene with each other by means of Southern blot analysis." (Van de Ven et al., 1987). The furin protein is the fur gene product and is expressed in certain normal tissues, as well as in specific types of tumors, for example, non-small cell lung tumors, mammary and colon carcinomas, urogenital tumors and hematologic malignancies. Recombinant DNA carrying portions of the genetic information for furin has been produced and patented. In this patent antibodies against the furin protein for diagnostic use, were also described. (Van de Ven, et al., 1987). The N-terminal region of furin shares 50% amino acid-identity with the catalytic domain of kex2 protease. On this basis, Fuller, et al. (1989) have proposed that it is a candidate for a human prohormone-processing enzyme.
Despite years of searching for mammalian proprotein processing proteases that operate in vivo, these proteins have been elusive and the field has been confusing. The strategy employed by the inventors which is described in the following sections has led to success in identifying a mammalian protein, convertin, which shows partial homology to the catalytic modules of both kex2 and the related bacterial subtilisins, and also has similarities to furin.